Jatropha curcas processing method and products

ABSTRACT

A process for preparing a food or feed composition from  J. curcas  is disclosed. The method involves adding an acidified aqueous solution to  J. curcas  components, to a final pH of between 1 and 5, incubating the acidified mixture for a period for a period of at least 1 hour, and centrifuging the incubated mixture to separate the mixture into three physically distinct fractions: (i) a light, upper fraction containing oil, (ii) an aqueous fraction containing soluble acid-extracted components and breakdown products, and (iii) a substantially detoxified solid cake which forms or is used in forming the food or feed composition. The acidified aqueous solution added may be acidified olive vegetation water having a ratio of hydroxytyrosol to oleuropein of between 5:1 to 100:1. Also disclosed are a food or feed composition, and oil and aqueous fractions formed by the method.

FIELD OF THE INVENTION

The present invention relates to a method for processing Jatropha curcasplants and products formed by the processing method.

BACKGROUND OF THE INVENTION

Jatropha curcas L. is a multipurpose shrub of significant economicimportance because of its several potential industrial and medicinaluses. Jatropha curcas L. or physic nut (or purging nut) is a droughtresistant large shrub or small tree, belonging to the genusEuphorbiaceae, producing oil containing seeds. The species has itsnatural distribution area in the Northeastern part of South America.(Heller, 1996) and central Africa and several countries in Asia. Theseeds of physic nut are a good source of oil, which can be used as adiesel substitute. They are used also in medicines, and soap andcosmetics manufacture in various tropical countries.

The fruit of J. curcas is green/yellow when fresh and contains seed. Theseed and seed products of J. curcas are potentially a source of highnutritional value, e.g., as animal feed. The levels of essential aminoacids, except lysine, in the seed cake are higher than that of theFAO/WHO reference protein for a five year old child in all the mealsamples on a dry matter basis. The major fatty acids found in the oilsamples were oleic (41.5-48.8%), linoleic (34.6-44.4%), palmitic(10.5-13.0%) and stearic (2.3-2.8%) acids. The residual protein-richseed cake, remaining after extraction of the oil, could form aprotein-rich ingredient in feeds for poultry, pigs, cattle and even fishif it could be detoxified.

Like the oil, the seed cake is toxic and therefore only suitable asanimal feed after processing. The toxicity of J. curcas is based onseveral components (phorbol esters, curcains, trypsin inhibitors andothers) which make complete detoxification a complicated process.Detoxification has been successful at laboratory scale (Gross et al., 161997; Martinez Herrera et al., 2006), but since the process iscomplicated, it is not suitable for small scale and local use. Largescale feed production, however, has to compete on a global market withhigh quality demands. Therefore, detoxification must be complete,constant and guaranteed, and is thus expected to be expensive. Hence, asuccessful penetration of J. curcas seed cake as feed to the market at aprofitable price seems doubtful.

Toxic components. The main toxic components are phorbol esters, althoughin Mexico accessions without, or with low content of phorbol esters havebeen found (Rivera Lorca & Ku Vera, 1997; Martinez Herrera et al., 2006;Basha & Sujatha, 2007). The seed cake of this so called ‘non’ or ‘low’toxic variety might be suitable for use as animal feed, but it stillcontains minor quantities of toxic components and resistance on the feedmarket towards this product is to be expected.

On the other hand, the seed cake is nutrient rich and therefore verysuitable as fertilizer (Table 3). Together with the fruit coats, themajor part of the nutrients can be recycled. When no fertilizers areused, which is assumed to be the case in the use of J. curcas as a lowinput crop, this recycling is necessary to maintain soil fertility,especially on non fertile marginal lands. Patolia (2007a) reported totalabove ground dry matter increase of 24% after 2 years.

Because of unavoidable inefficiencies, recycling nutrients will only beeffective at a certain production level that allows a high dynamicnutrient cycle to take place. Initiating a plantation on low or nonfertile soils therefore implies the need to use other fertilizers, atleast at the start, to boost crop growth and seed production in theinitial stages. The harvested part of J. curcas is the fruit, mostlycontaining three seeds. The seeds make up about 70% of the total weightof the fruit (30% fruit coat); the mature fruits have a moisture contentof circa 15%, the seeds circa 7%. The oil is stored in the interior ofthe seed: the kernel, which makes up circa 65% of the total mass of theseed. The moisture contents are circa 10% for the hull and circa 5% forthe kernel.

Oil Fraction and Quality.

The seed of J. curcas contains a viscous oil, highly suitable forcooking and lighting by itself and for the production of biodiesel. Thetotal fraction of oil, fats and carbohydrates is circa 30 to 35% for theseed and, since 99% of the oil is stored in the kernel, circa 50 to 55%for the kernel (Table 1).

The oil contains very little other components and has a very goodquality for burning. Cetane number of J. curcas oil (23-41) is close tocottonseed (35-40) and better than rapeseed (30-36), groundnut (30-41)and sunflower (29-37) (Vaitilingom & Liennard, 1997). The toxicity of J.curcas is mainly based on phorbol esters and curcains, which give nopollution when burnt. The oil is also very suitable fortransesterification into biodiesel (Mohibbe Azam et al., 2005).

The absence of sulphur dioxide (SO₂) in exhaust from diesel engines runon J. curcas oil shows that the oil may have a less adverse impact onthe environment (Kandpal & Madan, 1995). As J. curcas oil has a higherviscosity than diesel oil (53 versus 8 cSt at 30 C), blending J. curcasoil up to 50% with diesel oil is advised for use in a CompressionIgnition (C.I.) engine without major operational difficulties (Pramanik,2003). Other publications mention much lower values for viscosity (17.1cSt at 30 C), which would reduce the necessary blending fraction ofdiesel oil (Akintayo, 2004), however, conventional engines can beoperated by blending biomethanol or bioethanol (with gasoline) orbio-diesel (with diesel) from 3-20%. Some report that J. curcas oilshould only be used as ignition accelerator (Forson et al., 2004).

Seed Cake.

Like the oil, the seed cake is toxic and therefore only suitable asanimal feed after processing. The toxicity of J. curcas is based onseveral components (phorbol esters, curcains, trypsin inhibitors andothers) which make complete detoxification complicated. Detoxificationhas been successful at laboratory scale (Gross et al., 16 1997; MartinezHerrera et al., 2006), but since the process is complicated, it is notsuitable for small scale and local use. Large scale feed production,however, has to compete on a global market with high quality demands.Therefore, detoxification must be complete, constant and guaranteed, andis thus expected to be expensive. Hence, a successful penetration of J.curcas seed cake as feed to the market at a profitable price ischallenging. The main toxic, but potentially medicinal, components arephorbol esters, although in Mexico accessions without, or with lowcontent of phorbol esters have been found (Rivera Lorca & Ku Vera, 1997;Martinez Herrera et al., 2006; Basha & Sujatha, 2007). The seed cake ofthis so called ‘non’ or ‘low’ toxic variety might be suitable for use asanimal feed, but it still contains minor quantities of toxic componentsand resistance on the feed market towards this product is to beexpected.

On the other hand, the seed cake is nutrient rich and therefore verysuitable as fertilizer. Together with the fruit coats, the major part ofthe nutrients can be recycled. When no fertilizers are used, which isassumed to be the casein the use of J. curcas as a low input crop, thisrecycling is necessary to maintain soil fertility, especially on nonfertile marginal lands. Patolia (2007a) reported total aboveground drymatter increase. Because of unavoidable inefficiencies, recyclingnutrients will only be effective at a certain production level thatallows a high dynamic nutrient cycle to take place. Initiating aplantation on low or non fertile soils therefore implies the need to useother fertilizers, at least at the start, to boost crop growth and seedproduction in the initial stages.

The by-products of J. curcas, such as fruit coats, seed hulls and theremaining de-oiled seed cake after pressing, may be used for organicfertilization, or for the production of more energy. Seed hulls can beburnt and the seed cake and fruit pulp can be used for the production ofbiogas by anaerobic fermentation (Lopez et al., 1997; Staubmann et al.,1997; Vyas & Singh, 2007). By burning, most nutrients will be lost, butafter fermentation, most nutrients will remain in the effluent that canstill be used as a fertilizer to recycle nutrients. To maintain J.curcas production at a sustainable level, it is important to be awarethat a huge amount of nutrients are removed if J. curcas byproducts areexploited for additional valorization. However, the range in thereported nutrient values only comes from a few sources (Table 3), withclear variation. This indicates that environmental and managementconditions have a large effect on the eventual nutrient content of thevarious plant parts. Soil organic matter content decreases in aproduction system where nutrients are removed and not replenished byfertilization.

Oil Extraction.

For J. curcas oil extraction at small scale, various oil presses havebeen developed and modified from presses for other oil seed crops. Theyhave in common that they vary in design and are non-standardized, asthey were originally developed for other (edible) seeds and need to beoptimized for J. curcas seeds. Bielenberg Ram (Hand) Presses handle 7-10kg seed h-1 and spindle presses handle 15 kg seed h-1 (Mbeza et al.,2002). Commercially available pressing systems claim processing 500 kgseed h-1 (FIG. 15).

The recoverable oil fraction is clearly affected by pressing technology.For hand powered small scale pressing (such as the Bielenberg (Hand) RamPress), an oil yield of only 19% of the seed dry weight or 30% of thekernel was reported (Foidl & Eder, 1997; Augustus et al., 2002;Akintayo, 2004; Henning, 2004; Francis et al., 2005), which is about 60%of the total extractable amount. With mechanized pressing equipmentabout 75% of the oil can be recovered. Commercially available pressingsystems used for large-scale de-oiling of e.g. soybean and rapeseedreach up to 90%.

Modern extraction techniques can substantially raise the extractable oilfraction. Industrial extraction with organic solvents (mainly hexane)yield near 100% of the oil content, while extractions on water basis canyield from 65-97% of the oil, depending on, (a.o.) the composition ofthe extract solvent, the acidity (pH) and the temperature of the solvent(Shah et al., 2004; Shah et al., 2005).

Toxicity of the Cake.

A wide variation in toxic, but potentially medicinal, constituents, e.g.trypsin inhibitor in defatted kernels (18.4-27.5 mg g−1; Makkar et al.,1997) was observed, as well as a wide variation in saponins (1.8-3.4%;Makkar et al., 1997) and phytate (6.2-10.1%; Makkar et al., 1997).Phorbol esters are predominantly present, but are sometimes at lowlevels or not detected in provenances from Mexico. Phorbol ester contentranged from 0.87-3.32 mg g−1 of kernel weight in 17 provenances (Makkaret al., 1997; 3.85 mg g−1: Martinez Herrera et al., 2006).

Much attention to various aspects and tests of toxic components (phorbolesters and curcain) in J. curcas was reported at the ‘Jatropha 97’Symposium in Managua, Nicaragua (Chapter 4 in Gũbitz et al., 1997),including experiences for using proteins from toxic and ‘low toxic’ J.curcas seeds for livestock feed (Makkar & Becker, 1997). Toxicconstituents were found to be effective against a wide variety of pests(Solsoloy & Solsoloy, 1997; Rug & Ruppel, 2000). A 100% mortality ratewas obtained against mosquito (Culex quinque fasciatus Say), whenpetroleum extracts of J. curcas leaves were used as a larvicide(Karmegam et al., 1997). The toxicity of J. curcas is based on severalcomponents (phorbol esters, curcains, trypsin inhibitors and others)that are present in considerable amounts in all plant components(including the oil), which make complete detoxification a complicatedprocess.

Since the detoxification of J. curcas organic material is such acomplicated process, it has—so far—only been successful at laboratoryscale, and seems not to be suitable for small scale and localapplication. Like other J. curcas plant components, the seed cake istoxic and the prospect for successful penetration of the feed marketwith a detoxified product is challenging. The seed cake (either asremainder of the pressing process, or as a complete meal) is nutrientrich and therefore very suitable as fertilizer.

Phorbol esters of J. curcas decompose quickly as they are very sensitiveto elevated temperatures, light and atmospheric oxygen (NIH, 2007); theydecompose completely within 6 days (Rug & Ruppel, 2000).

To maintain J. curcas production at a sustainable level, it is importantto take notion of the huge amount of nutrients that are removed from thesoil if J. curcas by-products are exploited for additional uses,including the bio-refinery concept.

SUMMARY OF THE INVENTION

The invention includes, in one aspect, a process for preparing a food orfeed composition from J. curcas. The method includes the steps of:

(a) forming a mixture containing J. curcas components, with addition ofacid to a final pH of the mixture of between 1 and 5,

(b) incubating the mixture for a period of at least 1 hour, and

(c) centrifuging the incubated mixture to separate the slurry into threephysically distinct fractions: (i) a light, upper fraction containingoil, (ii) an aqueous fraction containing soluble acid-extractedcomponents and breakdown products, and (iii) a substantially detoxifiedsolid cake which forms or is used in forming the food or feedcomposition.

In one embodiment, step (a) in the method includes crushing J. curcas toform a slurry, and acidifying the slurry to a pH of between 1-5. Theslurry may be acidified by addition of acidified antioxidant solution.The acidified antioxidant solution may be added before, during, or aftercrushing the J. curcas components. The antioxidant solution may be olivevegetation water having ratio of hydroxytyrosol to oleuropein of between5:1 to 100:1. In certain embodiments, the olive vegetation watercomprises at least 0.1% (w/v) polyphenols. In other embodiments, theolive vegetation water comprises 5-10% (v/v) of an organic solvent.Preferred embodiments of organic solvents include methanol and ethanol.

In another embodiment, step (a) in the method includes crushing J.curcas to form a slurry, centrifuging the slurry to separate the slurryinto three physically distinct fractions: (i) a light, upper fractioncontaining oil, (ii) an aqueous fraction containing water-solublecomponents, and (iii) a first cake, and forming a cake slurry byaddition of an acidified aqueous solution to the first cake, at a pH ofbetween 1 and 5. The slurry may be formed by addition to the first cakeof acidified antioxidant solution. The antioxidant solution may be olivevegetation water having ratio of hydroxytyrosol to oleuropein of between5:1 to 100:1. In this embodiment, the light upper oil fraction from step(a) may be combined with the light upper oil fraction obtained in step(d), and the aqueous fraction from step (a) may be combined with theaqueous fraction obtained in step (d).

In still another embodiment, step (a) in the method includes adding anacidic aqueous solution to a first cake prepared from crushed J. Curcas,to form a cake slurry having a pH between 1-5. The cake slurry may beformed by addition to the first cake of acidified olive vegetation waterhaving ratio of hydroxytyrosol to oleuropein of between 5:1 to 100:1. Incertain embodiments, the olive vegetation water comprises at least 0.1%(w/v) polyphenols. In other embodiments, the olive vegetation watercomprises 5-10% (v/v) of an organic solvent. Preferred embodiments oforganic solvents include methanol and ethanol.

Acid or an acidic aqueous solution or acidified olive vegetation watermay be added to the cake components in step (a) to a final pH of between2-4, and an exemplary acidifying agent is a weak organic acid, such ascitric acid.

The incubating step (c) may be carried out at room temperature for aperiod of at least one day, for a period of at least 10 days, or for aperiod of at least 30 days or longer.

The process may further include extracting soluble components from theaqueous fraction obtained in step (c), and/or concentrating the aqueousfraction by removal of water.

In the preceding embodiments, the J. curcas components are selected fromthe fruit, the seed, or an already formed cake of J. curcas. Also in thepreceding embodiments, the olive vegetation water may comprise at least0.1% (w/v) polyphenols. In other embodiments, the olive vegetation watercomprises 5-10% (v/v) of an organic solvent. Preferred embodiments oforganic solvents include methanol and ethanol.

In another aspect, the invention includes a food or feed comprising J.curcas from which have been removed, toxic components that are extractedand/or degraded by incubation of components in an acidified aqueousslurry at pH 1-5 for at least one day.

The composition may be prepared by the methods disclosed above.

Also disclosed is an oil fraction from J. curcas formed by the steps of:

(a) pressing J. curcas components to form a cake and oil and aqueousfractions,

(b) after removing the oil and aqueous fractions, adding an acidifiedaqueous solution to the cake to form a slurry having a final pH ofbetween 1 and 5,

(c) incubating the slurry for a period for a period of at least 24hours,

(d) centrifuging the incubated slurry to separate the slurry into threephysically distinct fractions: (i) a light, upper fraction containingadditional oil, (ii) an aqueous fraction containing solubleacid-extracted components and breakdown products, and (iii) asubstantially detoxified solid cake which can be used as an animal feed,and

(e) isolating the light upper fraction obtained in step (d).

In one embodiment, step (a) includes adding the acidified antioxidantsolution before, during, or after pressing the J. curcas components. Inanother embodiment, step (b) may be carried out by adding to the cake,in forming a slurry, acidified olive vegetation water having ratio ofhydroxytyrosol to oleuropein of between 5:1 to 100:1. In certainembodiments, the olive vegetation water comprises at least 0.1% (w/v)polyphenols. In other embodiments, the olive vegetation water comprises5-10% (v/v) of an organic solvent. Preferred embodiments of organicsolvents include methanol and ethanol. The oil fraction may alsoincludes the oil fraction obtained in step (a).

Further disclosed is an aqueous fraction from J. curcas formed by thesteps of:

(a) pressing J. curcas components to form a cake and oil and aqueousfractions,

(b) after removing the oil and aqueous fractions, adding an acidifiedaqueous solution to the cake to form a slurry having a final pH ofbetween 1 and 5,

(c) incubating the slurry for a period for a period of at least 24hours,

(d) centrifuging the incubated slurry to separate the slurry into threephysically distinct fractions: (i) a light, upper fraction containingadditional oil, (ii) an aqueous fraction containing solubleacid-extracted components and breakdown products, and (iii) asubstantially detoxified solid cake which can be used as an animal feed,and

(e) isolating the aqueous fraction obtained in step (d).

The aqueous fraction may also include the aqueous fraction of step (a).The aqueous fraction may be further treated to extract medicinalcomponents therefrom. In one embodiment, step (a) includes adding theacidified antioxidant solution before, during, or after pressing the J.curcas components.

These and other objects and features of the invention will become morefully apparent when the following detailed description is read below.

In another aspect, provided herein is a method of extracting medicinalcompounds from J. curcas, comprising the steps of:

(a) pressing J. curcas components to form a cake and oil and aqueousfractions,

(b) removing the oil and aqueous fractions and then adding an aqueousacid solution to the cake to form a slurry having a final pH of between1 and 5,

(c) incubating the slurry for a period of at least 24 hours, and

(d) centrifuging the incubated slurry to separate the slurry into threephysically distinct fractions: (i) a light, upper fraction containingadditional oil, (ii) an aqueous fraction containing medicinal compoundsand breakdown products, and (iii) a substantially detoxified solid cake.

In one embodiment, step (a) additionally comprises pressing the J.curcas components in the presence of an aqueous acid solution. Inanother embodiment, the aqueous acid solution is an antioxidantsolution. In yet another embodiment, step (a) includes adding anacidified antioxidant solution before, during, or after the pressing ofthe J. curcas components. In certain embodiments, the antioxidantsolution is olive vegetation water. The olive vegetation water maycomprise at least 0.1% (w/v) polyphenols. The olive vegetation water mayhave a ratio of hydroxytyrosol to oleuropein of between 5:1 to 100:1.The olive vegetation water may comprise 5-10% (v/v) of an organicsolvent. In a preferred embodiment, the organic solvent is selected frommethanol and ethanol.

In another embodiment, the J. curcas components are selected from thefruit, the seed, or an already formed cake of J. curcas. In certainembodiments, the medicinal compounds are selected from curcin andphorbol esters.

DESCRIPTION OF THE INVENTION

In the present invention, acidulated water, also referred to as anacidic aqueous solution (e.g., citric acid 1%, chloridic acid 0.2 N orH₂SO₄ 0.2 N) may be used as a medium for extraction of hydrophobiccompounds present in the cake. Among these hydrophobic compounds aremost of the toxic compounds which make the cake poisonous. The aqueousextraction is carried at room temperature for few hours to several days.The suspension or slurry is then separated by a three phase centrifugesimilar to than commonly used by the olive oil industry.

Three phase centrifugation will produce a “light” phase represented bythe vegetable oil still trapped in the cake and thus recoverable by thisprocess, the “heavy” phase, represented by the aqueous fractioncontaining the majority of the hydrophilic compounds, which includesTrypsin inhibitors, sorbol esters and lecitins (saponins), and the solidfraction (cake).

There are three different embodiments contemplated. In the first, J.curcas components are crushed in the presence of an acidified aqueoussolution, to form a slurry, which is then incubated, e.g., 1 hour to 30days, to extract and/or detoxify soluble compounds from the J. curcascake components. After incubation, the slurry is centrifuged to form thethree fractions, all of which form various aspects of the invention: anupper oil phase, an intermediate aqueous fraction containing extractableproducts, e.g., medicinal products, and a lower, detoxified cake, whichmay be further processed into a food or feed composition. In certainexemplary methods, the acidified aqueous solution that is added to thecrushed J. curcas is an acidified olive vegetation water, that may behydroxytyrosol-rich, having a pH preferably between 1-5 and containing aratio of hydroxytyrosol to oleuropein of between 5:1 to 100:1. Asuitable hydroxytyrosol-rich composition is disclosed in co-owned U.S.U.S. Pat. No. 6,416,808, which is incorporated herein in its entirety.Exemplary methods of obtaining olive vegetation water are described inco-owned U.S. Pat. Nos. 6,165,475 and 6,197,308, each of which areexpressly incorporated herein by reference in their entirety. In certainembodiments and examples disclosed herein, the olive vegetation water isHIDROX® solution, an antioxidant solution prepared from olives.

In a second general embodiment, a J. curcas component slurry is firstcentrifuged to produce an upper oil fraction, an intermediate aqueousfraction and a lower cake. This initial step is preferably conductedunder relatively neutral-pH conditions, e.g., pH 5-8. The initial cakeis then further treated by addition of an acidified aqueous solution,e.g., the above acidified hydroxytyrosol-rich olive vegetation water, toform an acidified slurry, which is incubated as above, then centrifugedto form an upper oil fraction, an intermediate aqueous fraction, andlower, detoxified cake. The upper oil fraction may be combined with theinitial oil fraction, and the aqueous fraction may be combined with theinitial aqueous fraction. The aqueous fraction may be furtherconcentrated and/or used as a source of extractable medical or otherchemical components.

In a third general embodiment, an already formed J. curcas cake is usedas the starting material, and to this cake is added an acidified aqueoussolution, e.g., the above acidified hydroxytyrosol-rich olive vegetationwater, to form a cake slurry which is incubated as above, thencentrifuged to form an upper oil fraction, an intermediate aqueousfraction, and a lower, detoxified cake. In all of the precedingembodiments, the J. curcas components may be the fruit, the seed, or analready formed cake of J. curcas.

The presence of toxic/medicinal compounds in the aqueous fraction hasbeen confirmed by HPLC analysis. The toxicity of the residual cake hasbeen tested by animal toxicity studies conducted by BioQuant, Inc. SanDiego.

The aqueous extraction method has the advantage to:

(a) recover the residual oil trapped in the pressed cake.

(b) extract and separate the toxic components present in the cake whichare either hydrolyzed and/or are highly hydrophilic, and thus end up inthe water fraction, and

c) render the solid fraction less or totally non-toxic as confirmed byanimal studies.

Thus, the cake become a very valuable food and feed component which canbe formulated in a variety of foods for human and animals.

The aqueous fraction becomes a very valuable raw material for furtherextraction and isolation of compounds of chemical and pharmaceuticaluse, and can be further concentrated to reduce the content in water.This can be easily accomplished by common steam or vacuum evaporatorsgenerally used in the juice industry (orange juice) as an example andthen the water recycled for field irrigation of other uses in waterdeficient areas of the world. By performing the extraction of J. curcaswith an acidified antioxidant solution, the chemical compounds therebyextracted are protected from decomposition during the extraction,storage and concentration.

The concentrated juice can finally be sold as raw material for theextraction and separation of valuable compounds for medical, industrialand other uses based upon the active molecules present in or isolatedfrom the juice.

In one aspect, provided herein is a process for treating J. curcascomprising:

(a) forming a mixture containing J. curcas components, with addition ofacid to a final pH of the mixture of between 1 and 5,

(b) incubating the mixture for a period of at least 1 hour, and

(c) centrifuging the incubated mixture to separate the mixture intothree physically distinct fractions: (i) a light, upper fractioncontaining oil, (ii) an aqueous fraction containing solubleacid-extracted components and breakdown products, and (iii) asubstantially detoxified solid cake which forms or is used in formingthe food or feed composition.

In one embodiment, the process comprises the additional step: repeatingsteps (a)-(c).

In another embodiment, the process comprises the additional step: usingthe cake formed in step (c) as a food or feed composition.

In another embodiment of the process, step (a) includes crushing J.curcas components to form a slurry, and acidifying the slurry to a pH of1-5.

In another embodiment of the process, step (a) includes acidifying theslurry by adding an acidified antioxidant solution. In yet anotherembodiment, step (a) comprises adding an acidified antioxidant solutionbefore, during, or after crushing the J. curcas components. In stillanother embodiment, the antioxidant solution is olive vegetation water.In one embodiment, the olive vegetation water comprises at least 0.1%(w/v) polyphenols. In another embodiment, the olive vegetation watercomprises 5-10% (v/v) of an organic solvent.

In another embodiment of the process, step (a) includes crushing J.curcas components to form a slurry, centrifuging the slurry to separatethe slurry into three physically distinct fractions: (i) a light, upperfraction containing oil, (ii) an aqueous fraction containingwater-soluble components, and (iii) a first cake, and forming a cakeslurry by addition of an aqueous acid solution to the first cake, to apH of between 1 and 5. In some embodiments of the process, the aqueousacid solution is an antioxidant solution. In some embodiments, theantioxidant solution is olive vegetation water.

In another embodiment of the process, the light upper oil fraction fromstep (a) is combined with the light upper oil fraction obtained in step(c).

In another embodiment of the process, the aqueous fraction from step (a)is combined with the aqueous fraction obtained in step (c).

In another embodiment of the process, the mixture formed in step (a) hasa final pH of 2-4.

In another embodiment of the process, the mixture formed in step (a) isacidified by addition of a weak organic acid that imparts a final pH of2-4 to the slurry. In some embodiments, the weak organic acid is citricacid.

In another embodiment of the process, the incubating step (b) is carriedout at room temperature for a period of at least one day.

In another embodiment, the process further comprises extracting solublecomponents from the aqueous fraction obtained in step (c). In yetanother embodiment, the process further comprises concentrating theaqueous fraction by removal of water.

In another embodiment of the process, the olive vegetation watercomprises at least 0.1% (w/v) polyphenols. In yet another embodiment,the olive vegetation water has a ratio of hydroxytyrosol to oleuropeinof between 5:1 to 100:1. In still another embodiment, the olivevegetation water comprises 5-10% (v/v) of an organic solvent.

In another embodiment of the process, the J. curcas components areselected from the fruit, the seed, or an already formed cake of J.curcas.

In another aspect, provided herein is a food or feed compositionprepared according to the preceding process, and embodiments thereof.

In still another aspect, provided herein is an oil fraction obtainedaccording to the preceding process, and embodiments thereof. In oneembodiment, provided herein is the combined oil fractions of steps (a)and (c).

In yet another aspect, provided herein is an aqueous fraction obtainedaccording to the preceding process, and embodiments thereof. In oneembodiment, provided herein is the combined aqueous fractions of steps(a) and (c).

In one embodiment of the process, step (a) comprises:

(i) pressing J. curcas components to form a cake and oil and aqueousfractions, and(ii) removing the oil and aqueous fractions, and then adding an aqueousacid solution to the cake to form a slurry having a final pH of between1 and 5, and further comprising the step of: isolating the aqueousfraction obtained in step (c). In another embodiment, provided herein isthe aqueous fraction obtained according to the process. In oneembodiment, provided herein is the combined aqueous fractions of steps(a) and (c). In another embodiment, the aqueous fraction or fractionsare further treated to extract medicinal compounds therefrom.

In another embodiment of the process, step (a) comprises:

(i) pressing J. curcas components to form a cake and oil and aqueousfractions, and(ii) removing the oil and aqueous fractions, and then adding an aqueousacid solution to the cake to form a slurry having a final pH of between1 and 5, and further comprising the step of: isolating the light upperoil fraction obtained in step (c). In another embodiment, providedherein is the oil fraction obtained according to the process. In oneembodiment, provided herein is the combined oil fractions of steps (a)and (c).

In another aspect, provided herein is a method of extracting compoundsfrom J. curcas, comprising the preceding processes and embodimentsthereof. In one embodiment, the compounds are selected from curcin andphorbol esters.

Experimental

I. Jathropa Curcas Processing from Seed

Procedure A: To 200 kg seeds, prior to crushing, add the followingsolution A, made of 100 liters of 1% Citric Acid. Mix thoroughly to havea loose slurry and pour the mix onto a grinding machine. Grind mix intoa wet pulp and pump slurry into kneading tank. Stir for about 1 hour at30° C. Pump slurry into a three phase decanter and separate the threecomponents, Solid pulp, oil and aqueous extract. Examine threecomponents accordingly and calculate yields in oil. Save the solidfraction in freezer, until toxicity test is performed. Analyze aqueousfraction by HPLC.

Procedure B: To 200 kg seeds, prior to crushing, add the followingsolution B, made of 100 liters of 0.5% polyphenols extracted from thepulp of the olives in 1% citric acid. Mix thoroughly to obtain a slurryand proceed as above.

Procedure C: 200 kg seeds are processed without any addition of liquid.The solution A is added after the seeds are crushed into a thick pasteand pumped into a tank for 1 hr. kneading. Proceed then as above in 1and 2.

Procedure D: 200 kg seeds are processed without addition of any liquid.The solution B is added after the seeds are crushed into a thick pasteand pumped into a tank for 1 hr. kneading. Proceed then as above in 1and 2.

Procedure E (Control experiment): One kilogram of seeds are processed ina blender with addition of 500 ml water. The slurry is left at roomtemperature for 1.5 hrs and then centrifuged to separate liquid fractionfrom solid residue. Liquid is collected separately and analyzed by HPLC.The samples are frozen until further analysis is performed.

II. Processing from Solid Seed Cake

Procedure AI: To 200 kg dry cake add the following solution A, made of100 liters of 1% Citric Acid directly into kneading tank. Stir for about1.5 hour at 30° C. Pump slurry into a three-phase decanter and separatethe three components: solid pulp, crude oil and aqueous extract. Examinethree components accordingly and calculate yields in crude oil. Save thesolid fraction in freezer until toxicity test is performed. Analyzeaqueous fraction by HPLC.

Procedure BI: To 200 kg dry cake add the following solution B. made of100 liters of 0.5% polyphenols extracted from the pulp of the olives in1% citric acid. Mix thoroughly to obtain a slurry in kneading tank for1.5 hrs at 30° C. and proceed as above.

Procedure E2 (Control experiment): One kilogram of dry seed cake isprocessed in a blender with addition of 500 ml water. The slurry is leftat room temperature for 1.5 hrs and then centrifuged to separate liquidfraction from solid residue. Liquid is collected separately and analyzedby HPLC. The samples are frozen until further analysis is performed.

III. HPLC Jatropha Curca Processing and Detoxification.

I. HIDROX® 0.5% Liquid as antioxidant solution containing olivepolyphenols (e.g., hydroxytyrosol) was obtained from Creagri, Inc.(Hayward, Calif.). The HPLC profile of HIDROX® 0.5% liquid ischaracterized by the presence of a large peak (RT=5 m) corresponding tohydroxytyrosol (HT) with a percent area of approximately 40% of total UVabsorbing materials (Total Polyphenols, TP). A second small peak (RT=9.3min.) corresponds to tyrosol. The area is approximately 10% of the HTarea, 4% of total polyphenols (TP). The HPLC profile is thencharacterized by the presence of late peaks (at least 4-5) that elute athigh concentration of methanol in Buffer A (RT from 19.5 m to 20.8 m).These peaks correspond to oleuropein, verbascoside and their aglyconderivatives, which contribute all together to 46-47% of the TP. Total UVarea=41.5 million units.

2. Sample #1: Jatropha Curcas seeds (from Ghana) processed in thepresence of 1% citric acid solution: The peaks of these chromatogramscorrespond to 100% compounds derived from the Jatropha Curcas (JC) andsoluble in water (hydrophilic fraction). The front part of the spectrumis characterized by the presence of a large peak (RT=2 m) representingca. 16-17% of the total UV areas, in a possible concentration of ca.0.25% in weight of the total compounds in the solution (as directcomparison with 0.5% HIDROX® liquid). In addition, there are threeadditional peaks of relevance: the first one elutes with RT=1.6 m(3.5%), the second one with RT=2.4 m (3.8%) and the third one withRT=3.0 m (8.2%). A second set of peaks (three detectable) elutes with RTbetween 19.2 m and 20.0 m with percent areas of 4.5%, 6.3% and 4.0%respectively. Finally a third set of peaks (with two predominant peaksat RT=21.5 m and 21.8 m) is visible with a total % area of 22% (11.5%and 11% respectively). Total UV area=15.5 million units.

3. Sample #2: Jatropha Curcas cake (from the same source in Ghana)processed with HIDROX® 0.5% instead of 1% citric acid: The spectrumshould contain the total compounds of #1 and #2 in a firstapproximation. The list of fast peaks eluting between RT=0 and RT=3.1 minclude the large peak for JC (RT=2.0 m) which represents 21.2% of thetotal UV absorbing material, the two peaks at 5 m and 9.4 m (HT andTyrosol (Ty) from HIDROX® 0.5%, the first representing HT (15.6%) andthe second at 9.5 m representing Ty (1.7%). Also visible are the severalpeaks with low RT and high RT. Total UV area=49 million units.Observations: The total concentration of JC cake material in to HIDROX®0.5% is approximately 8 million units in a total of 49 million units, orapproximately 20%, assuming that the compounds in HIDROX® 0.5% areneither consumed nor diluted. The increase percentage of the JC peak at2 m, (21.2%) vs. the HT peak area (15.6%), however seems to indicatethat more than 60-65% of the JC cake compounds contribute to the totalpeak area of the extract. (Reduction of HT area from 37% to 15.6%, or42% reduction). The Ty concentration is also reduced from 3.64% to1.76%, or 48% reduction). The 3 peaks from JC cake are now present in3.1%, 5.2% and 9.5%, which corresponds to an increase of 73% and 86%.

4. Sample #3: Jatropha Curcas seeds processed with HIDROX® 0.5%: TheHPLC profile shows the presence of both peaks from HIDROX® 0.5% and JC.Specifically, from HIDROX® 0.5%, is well visible the HT peak RT=5.1 m(23.4%) and the Ty peak RT=9.4 m (2.1%). From the JC we clearly detectthe peak at RT=2.0 m (7%) and the 3 additional peaks at RT=1.7 m (2.3%),RT=2.4 m (3%) and RT=3 m (9.7%). Total area: 31.5 million units.

5. Conclusions: Extraction with an acidified aqueous solution or anaqueous EtOH (ethanol) solution (5%) seem to provide similar results.The extraction with the above solutions may results in detoxification ofboth the oil and the biomass in that:

-   -   (a) some of the compounds detected by HPLC analysis correspond        to phorbol esters (commercially available).    -   (b) the curcin (toxic protein) solubilizes in aqueous solutions.        In order to avoid oxidation of the above molecules in aqueous        solution, it is necessary to introduce an antioxidant component,        like hydroxytyrosol or other commercially available        antioxidants. The antioxidants will perform better if the        aqueous solution is acidified (citric acid or other organic and        non-organic acids). The pH we have used is ranging between 3.0        and 5.0. The detoxifying solution (water/antioxidant/acid and        possibly some percentage of EtOH (5%) can be added to the        Jatropha Curcas seeds prior to the milling and separation of the        oil from the biomass (cake), or can be used on the dry cake to        extract hydrophilic molecules and detoxify the biomass. Citric        acid alone does not seem to protect from oxidation as the        aqueous extract develops a strong odor after two-three months of        storage. Experiments conducted at laboratory scale and pilot        plant (200 kg seeds/cake) confirm the above. HPLC analysis of        samples of the resulting aqueous fraction indicate that ca.        70-80% of the compounds in the solution derive from the        extraction process. Subsequent use of the dry biomass as feed        for fish has confirmed the lack of toxicity of it.

IV. Quantization of HT (Hydroxytyrosol) in Freeze Dried Olive Juice byHPLC-Gradient

Equipment and Reagents:

HPLC grade methanol, ddH₂O, acetic acid and HIDROX® were used.

Standard Preparation:

Accurately dilute stock solution of standard (100 mg/2 ml HT; CaymanChemical) 1:3 with mobile phase (Solvent A) into a 2 ml micro tube. Mixwell. The working concentration of the standard is 1.67 mg/ml.

Sample Preparation:

Accurately weigh 100 mg+/−0.5 mg of sample and transfer to a 15 mlconical centrifuge tube. Add 10 ml of mobile phase (Solvent A) to thesample and mix well. Sonicate for 5 minutes then transfer 1 ml ofdissolved sample to a 2 ml micro tube. Centrifuge the 1 ml sample at11,000×g for 10 minutes. Remove all but the small pellet on the bottomto a new 2 ml micro tube.

Instrument Conditions:

-   -   Mobile Phase: (Solvent A): HPLC Grade ddH₂O with 5% HPLC Grade        Methanol and 3% HPLC Grade Acetic Acid (pH 2.7-2.8). (Solvent        B): 100% HPLC Grade Methanol    -   Flow Rate: 1.0 ml/min    -   Gradient: Solvent A (95.5%)/Solvent B (0.5%) isocratic for 20        min, then Solvent B 0.5-100% in 15 min.    -   Wavelength: OD 280 mm    -   Injection Volume: 20 μl    -   Column: Beckman Coulter Ultrasphere RP-C18 [4.6×150 mm]    -   Temperature: Column 20° C.+/−2° C.    -   Approximate Retention Times:        -   HT —5.9 minutes        -   Tyrosol—11.5 minutes

Procedure:

Mix 920 ml of HPLC Grade ddH₂O with 50 ml HPLC Grade Methanol and 30 mlHPLC Grade Acetic Acid “Solvent A”). Filter Solvent A with vacuum usinga 0.45 micron Nalgene Filter. Condition the analytical column for 30minutes before beginning calibration.

System Suitability:

Prepare a standard solution by thawing (from −20° C. freezer) a stockHIDROX® solution (1.67 mg/ml). Once thawed, the standard is discarded.Inject the standard solution to demonstrate presence of HT, retentiontime, peak area, peak height, and plate number. Inject the standardsolution 4 times to calibrate and establish the precision of thechromatographic system. Compute the relative standard deviation (% rsd)of the peak areas for HT. The system is considered suitable for assay ifthe % rsd of the four standard injections is <2%. As a further guide inassessing column performance, the column should develop ˜9000theoretical plates and the tailing factor should be less than 1.5. Atthe completion of the analysis, inject the standard solution as acalibration check. The calibration check should be +/−2% of the expectedconcentration.

Calculation:

The concentration of HT is calculated as follows:

Asp/As×S×p×V×Ws=mg/g, wherein:

-   -   Asp=Area of sample peak    -   As=Area of standard peak    -   S=working standard concentration in mg/ml    -   P=purity of standard    -   V=Sample Volume    -   Ws=Sample Weight

1-69. (canceled)
 70. An oil fraction from J. curcas formed by the stepsof: (a) crushing or grinding one or more J. curcas components to form aslurry, wherein the one or more J. curcas components is a J. curcasplant part selected from the group consisting of leaves, hulls, fruit,or seeds or pre-formed cake thereof; (b) acidifying the slurry of step(a) to a pH of 1-5 by adding an acidified aqueous solution to form anacidified slurry; (c) incubating the acidified slurry for a period of atleast 1 hour; (d) separating the incubated acidified slurry bycentrifuging or decanting into three distinct fractions: (i) a fractioncontaining oil, (ii) an aqueous fraction comprising unoxidized phorbolesters and curcin, and (iii) a detoxified solid cake; and (e) isolatingthe oil fraction in step (d).
 71. The oil fraction of claim 70, whereinthe acidified aqueous solution contains citric acid.
 72. The oilfraction of claim 70, wherein the acidified aqueous solution contains 1%citric acid.
 73. The oil fraction of claim 70, wherein the acidifiedaqueous antioxidant solution is olive vegetation water.
 74. The oilfraction of claim 73, wherein the olive vegetation water has a ratio ofhydroxytyrosol to oleuropein of between 5:1 to 100:1.
 75. The oilfraction of claim 73, wherein the olive vegetation water furthercomprises 5-10% (v/v) of an organic solvent.
 76. The oil fraction ofclaim 70, wherein the fraction containing oil is removed from thedetoxified solid cake and aqueous fraction, and the detoxified cake andaqueous fraction is further separated to provide three physicallydistinct fractions.
 77. The oil fraction of claim 70, wherein thefraction containing oil, the detoxified solid cake and the aqueousfraction are simultaneously separated from each other.
 78. The oilfraction of claim 70, wherein the acidified slurry is acidified to a pHof 2-4.
 79. A detoxified solid cake fraction from J. curcas formed bythe steps of: (a) crushing or grinding one or more J. curcas componentsto form a slurry, wherein the one or more J. curcas components is a J.curcas plant part selected from the group consisting of leaves, hulls,fruit, or seeds or pre-formed cake thereof; (b) acidifying the slurry ofstep (a) to a pH of 1-5 by adding an acidified aqueous solution to forman acidified slurry; (c) incubating the acidified slurry for a period ofat least 1 hour; (d) separating the incubated acidified slurry bycentrifuging or decanting into three distinct fractions: (i) a fractioncontaining oil, (ii) an aqueous fraction comprising unoxidized phorbolesters and curcin, and (iii) a detoxified solid cake; and (e) isolatingthe detoxified solid cake fraction in step (d).
 80. The detoxified solidcake fraction of claim 79, wherein the acidified aqueous solutioncontains citric acid.
 81. The detoxified solid cake fraction of claim79, wherein the acidified aqueous solution contains 1% citric acid. 82.The detoxified solid cake fraction of claim 79, wherein the acidifiedaqueous antioxidant solution is olive vegetation water.
 83. Thedetoxified solid cake fraction of claim 82, wherein the olive vegetationwater has a ratio of hydroxytyrosol to oleuropein of between 5:1 to100:1.
 84. The detoxified solid cake fraction of claim 82, wherein theolive vegetation water further comprises 5-10% (v/v) of an organicsolvent.
 85. The detoxified solid cake fraction of claim 79, wherein thefraction containing oil is removed from the detoxified solid cake andaqueous fraction, and the detoxified cake and aqueous fraction isfurther separated to provide three physically distinct fractions. 86.The detoxified solid cake fraction of claim 79, wherein the fractioncontaining oil, the detoxified solid cake and the aqueous fraction aresimultaneously separated from each other.
 87. The detoxified solid cakefraction of claim 79, wherein the acidified slurry is acidified to a pHof 2-4.